1. Field of the Invention
The present invention relates generally to the fields of molecular biology. More particularly, it concerns methods for the culture and production of mast cells from pluripotent cells.
2. Description of Related Art
Mast cells are cells found in mucosal and connective tissues throughout the body. Mast cells contain many granules rich in histamine and heparin and play a critical role in allergic responses. Mast cells are also involved in protective functions such as wound healing and defense against pathogens. Mature mast cells express high affinity receptors (FcεR1) which can bind IgE monomers. Antigen binding to this IgE triggers mast-cell degranulation and mast cell activation, producing a local or systemic immediate hypersensitivity reaction.
Mast cells can participate in certain inflammatory diseases and cancers. For example, mast cells can secrete proinflammatory cytokines involved in neuro-inflammatory processes and cancer. Mast cells can also accumulate in the stroma surrounding certain tumors such as adenocarcinoma, secreting molecules that can benefit tumors (Conti et al., 2007).
Isolation of mast cells is difficult due to the relatively low abundance and distribution of mast cells in a wide variety of tissues. Because of the difficulty in obtaining human mast cells in sufficient number and with high purity, previous studies largely relied on using rodent mast cells such as rat peritoneal mast cells or mouse bone marrow-derived cultured mast cells; however, rodent cells are not ideal because mast cells are heterogeneous, and there are many differences between species.
Nonetheless, mast cells are economically valuable and important tools for research. Mast cells may be used to test drug toxicity, histamine or granule release responses, and immunology-related responses, such as allergy-related or immune-related responses to compounds. Mast cells can be useful for purposes including drug screening and pre-clinical testing of putative biologically active compounds, including both allergy medicines, such as antihistamines, and non-allergy compounds. A convenient source of mast cells would also be particularly useful for labs interested in researching the biology, structure, and/or function of mast cells.
Previously, mast cells have been cultured from multipotent hematopoietic cord blood cells. Schernthaner et al. (2001) presents data regarding expression of the CD2 antigen by neoplastic cells. In order to analyze the expression of CD2 on immature “nonneoplastic” mast cells, Schernthaner et al. cultures mast cells from CD34+ cord blood cells using a cell culture medium comprising stem cell factor and IL-6. Similarly, Lappalainen et al. (2007) produced mast cells from human peripheral blood-derived CD34+ cells by culturing the multipotent hematopoietic cells in stem cell factor and certain cytokines at various timepoints. Like Schernthaner et al., multipotent hematopoietic progenitor cells were used.
It is currently not clear whether or how approaches using multipotent cells might be used or incorporated into a method for culturing mast cells from other cell types, such as pluripotent cells. In particular, the biological profile of multipotent hematopoietic (CD34+) progenitor cells are distinct from pluripotent cells, and there exists a need for methods for differentiating mast cells from pluripotent cells. In contrast to pluripotent cells, cord blood and other hematopoietic progenitor cells have limited expandability. Thus, for example, additional amounts of cord blood would be needed for the continued production of mast cells. In contrast, the expandability of pluripotent cells or cell lines is theoretically endless.
Differentiation of stem cells into various cell lines is an unpredictable art, and multiple factors including how stem cells are produced and maintained can affect subsequent attempts to differentiate the cells. For example, the responses of pluripotent cells as compared to other cell types, such as cord blood, can vary widely. Multiple factors can even affect the generation of mast cells from multipotent cells. For example, the source of progenitor cells, the methodology used, the number of mast cells generated, the degree of their maturity, and their phenotype and responsiveness towards various stimuli vary greatly (Kambe et al., 2000; Dahl et al., 2002; Wang et al., 2006). Thus, there exists a clear need for methods for the culture and differentiation of pluripotent cells into mast cells, and such methods would beneficially facilitate studies in these areas of research.